GIPR-agonist compounds

ABSTRACT

The present invention relates to compounds having activity at the human glucose-dependent insulinotropic polypeptide (GIP) receptor. The present invention also relates to compounds having an extended duration of action at the GIP receptor. Such compounds may be useful in the treatment of diabetes, including type 2 diabetes mellitus (“T2DM”). Also, the compounds may be useful in the treatment of obesity.

The present invention relates to compounds having activity at the humanglucose-dependent insulinotropic polypeptide (GIP) receptor. The presentinvention also relates to compounds having an extended duration ofaction at the GIP receptor. Compounds may be useful in the treatment oftype 2 diabetes mellitus (“T2DM”). Also, the compounds may be useful inthe treatment of obesity.

Over the past several decades, the prevalence of diabetes has continuedto rise. T2DM is the most common form of diabetes, accounting forapproximately 90% of all diabetes. T2DM is characterized by high bloodglucose levels associated mainly with insulin resistance. The currentstandard of care for T2DM includes diet and exercise, treatment withoral medications, and injectable glucose-lowering drugs, includingincretin-based therapies such as GLP-1 receptor agonists. A variety ofGLP-1 receptor agonists are currently available for treatment of T2DM,although currently marketed GLP-1 receptor agonists are generallydose-limited by gastrointestinal side effects such as nausea andvomiting.

Subcutaneous injection is the typical route of administration for theavailable GLP-1 receptor agonists. When treatment with oral medicationsand incretin-based therapies are insufficient, insulin treatment isconsidered. Despite the advances in treatment available today, manypatients with T2DM are unable to reach their glycemic control goals.Uncontrolled diabetes leads to several conditions associated withincreased morbidity and mortality of patients.

There is a need for a treatment to enable more patients with T2DM toreach their glycemic treatment goals.

Obesity is a complex medical disorder resulting in excessiveaccumulation of adipose tissue mass. Today obesity is a global publichealth concern that is associated with undesired health outcomes andmorbidities. Desired treatments for patients with obesity should reduceexcess body weight, improve obesity-related co-morbidities, and maintainlong-term weight reduction. Available treatments for obesity areparticularly unsatisfactory for patients with severe obesity. There is aneed for alternative treatment options to induce therapeutic weight lossin patients in need of such treatment.

WO2016/111971 describes peptides stated to have GLP-1R and GIPR agonistactivities. WO2013/164483 also discloses compounds stated to have GLP-1Rand GIPR activities.

WO2018/181864 discloses compounds stated to have GIPR agonist activity.

There is a need for T2DM treatments capable of providing effectiveglucose control for a larger portion of the patients in need of suchtreatment. There is a further need for T2DM treatments capable ofproviding effective glucose control and with a favorable side effectprofile. There is a need for alternate treatment options to providetherapeutic weight loss in a patient in need of such treatment, such asa patient with severe obesity. There is a desire for diabetes treatmentoptions that may be combined with insulin therapy and/or incretintherapy to provide the patient with superior glycemic outcomes and/ormore desirable side effect profiles.

Compounds with extended duration of action at the GIP receptor aredesirable to allow for less frequent dosing of the compound.

Accordingly, embodiment 1 is a compound of Formula IZ₁X₁X₂EGTX₆ISDYSIX₁₃LDX₁₆X₁₇X₁₈QX₂₀X₂₁X₂₂VX₂₄X₂₅X₂₆LX₂₈X₂₉GPSSGAPPPSZ₂(SEQ ID NO:4) wherein:

-   -   Z₁ is a modification of the N-terminal amino group wherein the        modification is selected from the group consisting of acetyl and        absent;    -   X₁ is selected from the group consisting of Y and D-Tyr;    -   X₂ is selected from the group consisting of Aib, A, and D-Ala;    -   X₆ is selected from the group consisting of F, αMeF, Iva, L,        αMeL, and αMeF(2F);    -   X₁₃ is selected from the group consisting of αMeL, A, L, and        Aib;    -   X₁₆ is selected from the group consisting of K, E, and Orn;    -   X₁₇ is selected from the group consisting of I and        K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)_(q)—CO₂H;    -   X₁₈ is selected from the group consisting of H, A, and    -   X₂₀ is selected from the group consisting of Aib and Q;    -   X₂₁ is selected from the group consisting of D and E;    -   X₂₂ is selected from the group consisting of F and αMeF;    -   X₂₄ is selected from the group consisting of E, N, Q, and D-Glu;    -   X₂₅ is selected from the group consisting of Y, 4-Pal, W, and        αMeY;    -   X₂₆ is selected from the group consisting of L and        K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)_(q)—CO₂H;    -   X₂₈ is selected from the group consisting of E and A;    -   X₂₉ is selected from the group consisting of G, A, Q, and T; q        is selected from the group consisting of 16 and 18; and    -   Z₂ is absent or a modification of the C-terminal group, wherein        the modification is amidation;    -   wherein one and only one selected from X₁₇ and X₂₆ is        K(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)_(q)—CO₂H;        or a pharmaceutically acceptable salt thereof.

An embodiment 2 provides a compound, or a pharmaceutically acceptablesalt thereof, of embodiment 1 wherein Z₁ is absent and X₁ is Y.

An embodiment 3 provides a compound, or pharmaceutically acceptable saltthereof, of embodiment 1 or embodiment 2 wherein X₂ is Aib.

An embodiment 4 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 3 wherein:

X₆ is selected from the group consisting of F and αMeF(2F).

An embodiment 5 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 4 wherein:

X₁₃ is selected from the group consisting of L and αMeL.

An embodiment 6 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 5 wherein X₁₆ is K or Orn.

An embodiment 7 provides a compound, or a pharmaceutically acceptablesalt thereof, of embodiment 6 wherein X₁₆ is K.

An embodiment 8 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 7 wherein X₁₈ is H.

An embodiment 9 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 8 wherein X₂₀ is Aib; andX₂₂ is F.

An embodiment 10 provides a compound, or a pharmaceutically acceptablesalt thereof, as claimed of any one of embodiments 1 to 9 wherein X₂₁ isD.

An embodiment 11 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 10 wherein X₂₅ is 4-Pal orY.

An embodiment 12 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 11 wherein:

X₁₇ isK(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)_(q)—CO₂H; and

X₂₆ is L.

An embodiment 13 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 11 wherein:

X₁₇ is I; and

X₂₆ isK(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)_(q)—CO₂H.

An embodiment 14 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 13 wherein q is 16.

An embodiment 15 provides a compound, or a pharmaceutically acceptablesalt thereof, of any one of embodiments 1 to 13 wherein q is 18.

An embodiment 16 provides a compound, or a pharmaceutically acceptablesalt thereof, of embodiment 1 wherein: Z₁ is absent;

X₁ is Y;

X₂ is selected from the group consisting of Aib and D-Ala, X₆ is F;

X₁₃ is selected from the group consisting of αMeL and L;

X₁₆ is selected from the group consisting of K and Orn;

X₁₈ is selected from the group consisting of H and A;

X₂₀ is Aib;

X₂₂ is F;

X₂₄ is selected from the group consisting of E, N, and D-Glu;

X₂₅ is selected from the group consisting of Y, 4-Pal, and W;

X₂₈ is selected from the group consisting of E and A;

X₂₉ is selected from the group consisting of G, A, and Q; and q isselected from the group consisting of 16 and 18.

An embodiment 17 provides a compound, or a pharmaceutically acceptablesalt thereof, of embodiment 16 wherein:

Z₁ is absent;

X₂ is Aib;

X₁₃ is αMeL;

X₁₈ is H;

X₂₄ is selected from the group consisting of E and D-Glu;

X₂₅ is selected from the group consisting of Y and 4-Pal;

X₂₈ is E;

X₂₉ is selected from the group consisting of G and A.

An embodiment 18 provides a compound, or a pharmaceutically acceptablesalt thereof, of embodiment 17 wherein: X₁₇ isK(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)_(q)—CO₂H; and

X₂₆ is L.

An embodiment 19 provides a compound, or a pharmaceutically acceptablesalt thereof, of embodiment 17 wherein: X₁₇ is I; and X₂₆ isK(2-[2-(2-amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)_(q)—CO₂H.

An embodiment 20 provides a compound, or a pharmaceutically acceptablesalt thereof, of embodiment 1 wherein the compound is selected from thegroup consisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO: 10, and SEQ ID NO:11.

An embodiment 21 provides a compound or pharmaceutically acceptable saltthereof, of embodiment 1 wherein the compound is SEQ ID NO:5. Anembodiment 22 provides a compound or pharmaceutically acceptable saltthereof, of embodiment 1 wherein the compound is SEQ ID NO:6. Anembodiment 23 provides a compound or pharmaceutically acceptable saltthereof, of embodiment 1 wherein the compound is SEQ ID NO:7. Anembodiment 24 provides a compound or pharmaceutically acceptable saltthereof, of embodiment 1 wherein the compound is SEQ ID NO:8. Anembodiment 25 provides a compound or pharmaceutically acceptable saltthereof, of embodiment 1 wherein the compound is SEQ ID NO:9. Anembodiment 26 provides a compound or pharmaceutically acceptable saltthereof, of embodiment 1 wherein the compound is SEQ ID NO:10. Anembodiment 27 provides a compound or pharmaceutically acceptable saltthereof, of embodiment 1 wherein the compound is SEQ ID NO:11.

An embodiment provides a method of treating a condition selected fromthe group consisting of diabetes, obesity, and metabolic syndrome,comprising administering to a subject in need thereof, an effectiveamount of a compound of any one of embodiments 1 to 27, or apharmaceutically acceptable salt thereof. An embodiment provides amethod of treating a condition selected from the group consisting ofT2DM, obesity, and metabolic syndrome, comprising administering to asubject in need thereof, an effective amount of a compound of Formula Ior a pharmaceutically acceptable salt thereof. An embodiment provides amethod for providing therapeutic weight loss comprising administering toa subject in need thereof, an effective amount of a compound of FormulaI, or a pharmaceutically acceptable salt thereof. An embodiment is amethod of treating T2DM comprising administering to a subject in needthereof, and effective amount of a compound of any one of embodiments 1to 27, or a pharmaceutically acceptable salt thereof.

An embodiment provides a compound of Formula I, or a pharmaceuticallyacceptable salt thereof; for use in therapy. An embodiment provides acompound of any one of embodiments 1 to 27, or a pharmaceuticallyacceptable salt thereof, for use in therapy to treat a conditionselected from the group consisting of diabetes, obesity, and metabolicsyndrome. An embodiment provides a compound of Formula I, or apharmaceutically acceptable salt thereof, for use in therapy to treat acondition selected from the group consisting of T2DM, obesity, andmetabolic syndrome. In an embodiment, the condition is T2DM. In anembodiment, the condition is obesity. In an embodiment, the condition istype 1 diabetes (T1DM). In an embodiment, the condition is diabetes in apatient receiving insulin therapy. In an embodiment, the condition ismetabolic syndrome.

The compounds of Formula I, or a pharmaceutically acceptable saltthereof, may be useful in the treatment of a variety of symptoms ordisorders. For example, certain embodiments provide a method fortreatment of T2DM in a patient comprising administering to a subject inneed of such treatment an effective amount of a compound of Formula I,or a pharmaceutically acceptable salt thereof. In an embodiment is amethod for treatment of obesity in a patient comprising administering toa subject in need of such treatment an effective amount of a compound ofFormula I, or a pharmaceutically acceptable salt thereof. In anembodiment, the method is inducing non-therapeutic weight loss in asubject, comprising administering to a subject in need of such treatmentan effective amount of a compound of Formula I, or a pharmaceuticallyacceptable salt thereof.

In certain embodiments, the present invention provides a method fortreatment of metabolic syndrome in a patient comprising administering toa subject in need of such treatment an effective amount of a compound ofFormula I, or a pharmaceutically acceptable salt thereof. In anembodiment, the method is treatment of diabetes in a patient receivinginsulin treatment, comprising administering to a subject in need of suchtreatment an effective amount of a compound of Formula I, or apharmaceutically acceptable salt thereof. Also provided herein is acompound of the present invention for use in simultaneous, separate andsequential combinations with one or more agents selected from metformin,a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor,a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, a growthdifferentiation factor 15 modulator (“GDF15”), a peptide tyrosinetyrosine modulator (“PYY”), a modified insulin, amylin, a dualamylin-calcitonin receptor agonist, and an oxyntomodulin agonist (“OW”)in the treatment of a condition selected from the group consisting ofT2DM, obesity, and metabolic syndrome. Also provided herein is acompound of the present invention for use in simultaneous, separate, andsequential combinations with one or more agents selected from metformin,a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor,a sodium-glucose cotransporter-2 (SGLT-2) inhibitor, a growthdifferentiation factor 15 modulator (“GDF15”), a peptide tyrosinetyrosine analog (“PYY”), a modified insulin, an amylin receptor agonist,a dual amylin-calcitonin receptor agonist, a modified urocortin-2(UCN-2) analog, a glucagon-like-peptide-1 (GLP-1) receptor agonist, aglucagon receptor agonist, and a dual GLP-1-glucagon receptor agonistincluding oxyntomodulin and analogs thereof, in the treatment of acondition selected from the group consisting of T2DM, obesity, andmetabolic syndrome. In an embodiment, a compound of the presentinvention is provided in a fixed dose combination with one or moreagents selected from metformin, a thiazolidinedione, a sulfonylurea, adipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, PYY, amodified insulin, amylin, a dual amylin-calcitonin receptor agonist, andOXM. In an embodiment, a compound of the present invention is providedin a fixed dose combination with one or more agents selected frommetformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4inhibitor, a SGLT-2 inhibitor, GDF15, a PYY analog, a modified insulin,an amylin receptor agonist, a dual amylin-calcitonin receptor agonist, amodified urocortin-2 (UCN-2) analog, a glucagon-like-peptide-1 (GLP-1)receptor agonist, a glucagon receptor agonist, and a dual GLP-1-glucagonreceptor agonist including OXM and analogs thereof. In an embodiment isa compound of the present invention for use in simultaneous, separateand sequential combinations with one or more agents selected frommetformin, a thiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4inhibitor, a SGLT-2 inhibitor, GDF15, PYY, a modified insulin, amylin, adual amylin-calcitonin receptor agonist, and OXM in the treatment of acondition selected from the group consisting of T2DM and obesity. In anembodiment is a compound of the present invention for use insimultaneous, separate and sequential combinations with one or moreagents selected from metformin, a thiazolidinedione, a sulfonylurea, adipeptidyl peptidase 4 inhibitor, a SGLT-2 inhibitor, GDF15, a PYYanalog, a modified insulin, an amylin receptor agonist, a dualamylin-calcitonin receptor agonist, a modified urocortin-2 (UCN-2)analog, a glucagon-like-peptide-1 (GLP-1) receptor agonist, a glucagonreceptor agonist, and a dual GLP-1 glucagon receptor agonist includingOXM and analogs of, in the treatment of a condition selected from thegroup consisting of T2DM and obesity. In an embodiment is a compound ofthe present invention for use in simultaneous, separate and sequentialcombinations with one or more agents selected from metformin, athiazolidinedione, a sulfonylurea, a dipeptidyl peptidase 4 inhibitor,and a SGLT-2 inhibitor in the treatment of a condition selected from thegroup consisting of T2DM and obesity.

In an embodiment is a method for treating diabetes in a patientreceiving insulin therapy, comprising administering an effective amountof a Compound of Formula I or a pharmaceutically acceptable saltthereof, to a patient in need thereof. An embodiment is treatment to apatient administered insulin therapy for T1DM. An embodiment istreatment to patient administered insulin therapy for T2DM. Anembodiment is once weekly dosing. An embodiment is subcutaneoustreatment once weekly to a patient administered insulin therapy. Anembodiment exists wherein the insulin therapy comprises basal insulintherapy. An embodiment exists wherein the insulin therapy comprisesmealtime insulin therapy. An embodiment exists wherein the insulintherapy comprises ultra-rapid insulin therapy. Insulin therapyadministered with acute infusions of a compound of Formula I, or apharmaceutically acceptable salt thereof, may enhance glucagon excursionin patients undergoing hypoglycemic clamp, thus enhancing the body'snatural defense against hypoglycemia. A compound of Formula I, or apharmaceutically acceptable salt thereof, can be dosed once weeklyindependent of the type of insulin used or doses of insulin used. Anembodiment is a compound of any one of embodiments 1 to 27, or apharmaceutically acceptable salt thereof, administered as an effectiveamount to a patient receiving insulin therapy, independent of the typeof insulin used or doses of insulin used. An embodiment is a compound ofany one of embodiments 1 to 27, or a pharmaceutically acceptable saltthereof, dosed once weekly as an effective amount to a patient receivinginsulin therapy independent of the type of insulin used or doses ofinsulin used. As used herein “insulin therapy” means treatment of apatient with diabetes using an approved insulin treatment. Such insulintherapy is known to the skilled artisan and/or clinical health careprofessional. For example, insulin therapy may comprise treatment usingbasal insulin. Such basal insulin “insulin therapy” may be used in adosing regimen with mealtime insulin and/or ultra-rapid insulin. As usedherein, “mealtime insulin” means insulin and/or modified insulin to beadministered with meals, for example, but not limited to, insulinlispro. As used herein, “basal insulin” means modified insulin with alonger duration of action, such as, for example, but not limited to,insulin glargine.

Another embodiment provides the use of a compound of Formula I, or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment of a condition selected from the groupconsisting of T2DM, obesity, and metabolic syndrome. An embodimentprovides the use of a compound of any one of embodiments 1 to 27, or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for the treatment of a condition selected from the groupconsisting of diabetes, obesity, and metabolic syndrome. In anembodiment, the medicament is for the treatment of T2DM. In anembodiment, the medicament is for the treatment of obesity. In anembodiment, the medicament s for use in the treatment of diabetes in apatient receiving insulin therapy.

Another embodiment provides a pharmaceutical composition comprising acompound of Formula I, or a pharmaceutically acceptable salt thereof,and at least one selected from the group consisting of a carrier,diluent, and excipient. In an embodiment, a pharmaceutical compositionfor subcutaneous administration is provided.

As used herein, the term “treating” or “to treat” includes restraining,slowing, stopping, or reversing the progression or severity of asymptom, condition, or disorder.

Certain compounds of the present invention are generally effective overa wide dosage range. For example, dosages for once weekly parenteraldosing may fall within the range of 0.05 mg to about 60 mg per personper week.

The compounds of Formula I, or a pharmaceutically acceptable saltthereof, include novel amino acid sequences having affinity for the GIPreceptor, with desired potency at the receptor. GIP is a 42 amino acidpeptide (SEQ ID NO:1), which, like GLP-1, is known as an incretin, andit plays a physiological role in glucose homeostasis by stimulatinginsulin secretion from pancreatic beta cells in the presence of glucose.

GLP-1 is a 36 amino acid peptide, the major biologically active fragmentof which is produced as a 30-amino acid, C-terminal amidated peptide(GLP-17-36) (SEQ ID NO:2).

Glucagon is a 29-amino acid peptide hormone (SEQ ID NO:3) secreted byα-cells of the islet of Langerhans in the pancreas and is involved inglucose homeostasis.

The compounds of present invention provide desired potency at the GIPreceptor with high degree of selectivity against the GLP-1R and theGlucagon receptor. In an embodiment, compounds have desired GIP receptoractivity with extended duration of action.

As used herein the term “amino acid” means both naturally occurringamino acids and unnatural amino acids. The amino acids are typicallydepicted using standard one letter codes (e.g., L=leucine), as well asalpha-methyl substituted residues of natural amino acids (e.g., α-methylleucine, or αMeL, and α-methyl phenylalanine, or αMeF) and certain otherunnatural amino acids, such as alpha-amino isobutyric acid, or “Aib,”“4Pal,” “Orn,” and the like. The structures of these amino acids appearbelow:

As used herein, “Orn” means L-ornithine. As used herein, “4Pal” means3-(4-Pyridyl)-L-alanine. As used herein, “αMeF(2F)” means alpha-methyl2-fluoro-L-phenylalanine. As used herein, “αMeY” and “αMeL” meanalpha-methyl-L-tyrosine and alpha-methyl-L-leucine, respectively. Asused herein, “e” and “D-Glu” mean D-glutamic acid. As used herein,“D-Tyr” and “y” each mean D-tyrosine. As used herein, “D-Ala” and “a”each mean D-alanine. As used herein, “αMeF” means alpha-methyl-F andalpha-methyl-Phe. As used herein “Iva” means L-isovaline.

In an embodiment, the conjugation is an acylation. In an embodiment, theconjugation is to the epsilon-amino group of the K side-chain. In anembodiment of the compounds of the present invention, a fatty acidmoiety is conjugated, via a linker, to a K at position 17. In anembodiment of the compounds of the present invention, a fatty acidmoiety is conjugated, via a linker, to a K at position 26.

In an embodiment, q is selected from the group consisting of 16 and 18.In an embodiment, q is 16. In an embodiment, q is 18.

When used herein in reference to the GIP receptor the terms “activity,”“activate[s]” “activat[ing]” and the like refers to the capacity of acompound, or a pharmaceutically acceptable salt thereof, to bind to andinduce a response at the receptor, as measured using assays known in theart, such as the in vitro assays described below.

The affinity of compounds of Formula I, or a pharmaceutically acceptablesalt thereof, for the GIP receptor may be measured using techniquesknown for measuring receptor binding levels in the art, including, forexample, those described in the examples below, and is commonlyexpressed as a K_(i) value. The activity of the compounds of the presentinvention at the GIP receptor may also be measured using techniquesknown in the art, including for example the in vitro activity assaysdescribed below, and is commonly expressed as an EC₅₀ value, which isthe concentration of compound causing half-maximal simulation in a doseresponse curve.

In addition, data is provided for each compound for activity andaffinity at the GLP-1 and glucagon receptors, to demonstrate the degreeof selectivity of the compounds of the present invention for the GIPR.

In an embodiment, a pharmaceutical composition of a compound of FormulaI is suitable for administration by a parenteral route (e.g.,subcutaneous, intravenous, intraperitoneal, intramuscular, ortransdermal). Some pharmaceutical compositions and processes forpreparing same are well known in the art. (See, e.g., Remington: TheScience and Practice of Pharmacy (D. B. Troy, Editor, 21st Edition,Lippincott, Williams & Wilkins, 2006)).

Compounds of the present invention may react with any of a number ofinorganic and organic acids/bases to form pharmaceutically acceptableacid/base addition salts. Pharmaceutically acceptable salts and commonmethodology for preparing them are well known in the art. (See, e.g., P.Stahl, et al. Handbook of Pharmaceutical Salts: Properties, Selectionand Use, 2nd Revised Edition (Wiley-VCH, 2011)). Pharmaceuticallyacceptable salts of the present invention include, but are not limitedto, sodium, trifluoroacetate, hydrochloride, ammonium, and acetatesalts. In an embodiment, a pharmaceutically acceptable salt is selectedfrom the group consisting of sodium, hydrochloride, and acetate salts.

The present invention also encompasses novel intermediates and processesuseful for the synthesis of compounds of the present invention, or apharmaceutically acceptable salt thereof. The intermediates andcompounds of the present invention may be prepared by a variety ofprocedures known in the art. In particular, the Examples below describea process using chemical synthesis. The specific synthetic steps foreach of the routes described may be combined in different ways toprepare compounds of the present invention. The reagents and startingmaterials are readily available to one of ordinary skill in the art.

When used herein, the term “effective amount” refers to the amount ordose of a compound of the present invention, or a pharmaceuticallyacceptable salt thereof, which, upon single or multiple doseadministration to the patient, provides the desired effect in thepatient under diagnosis or treatment. An effective amount can bedetermined by a person of skill in the art using known techniques and byobserving results obtained under analogous circumstances. In determiningthe effective amount for a subject, a number of factors are considered,including, but not limited to: the species of mammal; its size, age, andgeneral health; the specific disease or disorder involved; the degree ofor involvement or the severity of the disease or disorder; the responseof the individual patient; the particular compound administered; themode of administration; the bioavailability characteristics of thepreparation administered; the dose regimen selected; the use ofconcomitant medication; and other relevant circumstances.

When used herein, the term “subject in need thereof” refers to a mammal,preferably a human, with a disease or condition requiring treatment ortherapy, including for example those listed in the preceding paragraphs.

As used herein, “EDTA” means ethylenediatninetetraacetic acid. As usedherein; “DMSO” means dimethyl sulfoxide. As used herein, “CPM” meanscounts per minute. As used herein, “IBMX” means3-isobutyl-1-methylxanthine. As used herein, “LC/NIS” means liquidchromatography/mass spectrometry. As used herein, “HTRF” meanshomogeneous time-resolved fluorescence. As used herein, “DMF” refers toN,N-dimethylformamide. As used herein, “DCM” refers to dichloromethane.As used herein, “TFA” refers to trifluoroacetic acid. As used herein,“TFA salt” refers to trifluoroacetate salt. As used herein, “RP HPLC”means reversed-phase high performance liquid chromatography.

The invention is further illustrated by the following examples, whichare not to be construed as limiting.

PEPTIDE SYNTHESIS Example 1Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)AQ-Aib-DFVNWLLAQGPSSGAPPPS-NH₂(SEQ ID NO:5)

The structure of SEQ ID NO:5 is depicted below using the standard singleletter amino acid codes with the exceptions of residues D-Ala2, K17,Aib20, and Ser39, where the structures of these amino acid residues havebeen expanded:

The peptide backbone of Example 1 is synthesized usingfluorenylmethyloxycarbonyl (Fmoc)/tert-butyl (t-Bu) chemistry on aSymphony multiplex peptide synthesizer (Gyros Protein Technologies.Tucson, Ariz.; 3.3.0.1), software version 3.3.0.

The resin consists of aminomethyl polystyrene functionalized with a RinkAmide linker (polystyrene AM RAM, RAPP polymeric GmbH, H40023, 200-400mesh) at a substitution of 0.8 mmol/g, Standard side-chain protectinggroups are used with the following exceptions: Fmoc-Lys(Mtt)-OH, whereMtt is 4-methyltrityl, is used for the lysine at position 17 andBoc-Tyr(t-Bu)-OH is used for the tyrosine at position 1, Fmoc groups areremoved prior to each coupling step (2×10 minutes) using 20% piperidinein DMF. All standard amino acid couplings are performed using an equalmolar ratio of Fmoc amino acid (0.3 M in DMF), diisopropylcarbodiimide(0.9 M in DCM) and Oxyma (0.9 Min DMF) at a 9-fold molar excess over thetheoretical peptide loading. Couplings are allowed to proceed for 1.5hours, with the following exceptions: coupling of valine, 3 hours;coupling of Ca-methylated amino acids, 6 hours; coupling toCa-methylated amino acids, 6-10 hours. After completion of the synthesisof the peptide backbone, the resin is thoroughly washed with DCM toremove residual DMF. The Mtt protecting group on the lysine at position17 is selectively removed from the peptide resin using three treatmentsof 30% hexafluoroisopropanol (Oakwood Chemicals) in DCM (3×20-minutetreatment), and the resin is thoroughly washed with DCM and DMF.

Subsequent attachment of the fatty acid-linker moiety is accomplished bycoupling of 2-[2-(2-Fmoc-amino-ethoxy)-ethoxy]-acetic acid(Fmoc-AEEA-OH, ChemPep, Inc.) and Fmoc-glutamic acid α-t-butyl ester(Fmoc-Glu-OtBu, Ark Pharm, Inc.) following the procedures describedabove for standard coupling and deprotection reactions. After removal ofthe final Fmoc protecting group, mono-OtBu-eicosanedioic acid (WuXiAppTec, Shanghai, China) is coupled for 1 hour using a 4-fold excess ofthe diacid, PyBOP, and diisopropylethylamine (1:1:1 mol/mol/mol) in 1:1DCM/DMF.

After the synthesis is complete, the peptide-resin is washed with DCMand then thoroughly dried over vacuum. The dry peptide-resin is treatedwith cleavage cocktail (10 mL TFA, 0.5 mL triisopropylsilane, 0.5 mLwater, and 0.5 mL 1,2-ethanedithiol) for 2 hours at room temperature.The peptide resin solution is filtered into a 50-mL conical centrifugetube and treated with 5-fold excess volume of cold diethyl ether (−20°C.) to precipitate the crude peptide. The peptide/ether suspension iscentrifuged at 3000 rcf for 1.5 min, to form a solid pellet and thesupernatant is decanted. The pellet is washed further two times withcold diethyl ether, centrifuging for 1 min. each time, then dried invacuo. The crude peptide is solubilized in 20% acetic acid/80% water andpurified by RP-HPLC on a SymmetryPrep 7 μm C18 preparative column(18×300 mm, Waters) with linear gradients of 100% acetonitrile and 0.1%TEA/water buffer system (25-45% acetonitrile in 65 min). The purity ofpeptide is assessed using analytical RP-HPLC and pooling criteriais >95%, The main pool purity of compound of example 1 is found to be96.8%. Subsequent lyophilization of the final main product pool yieldedthe lyophilized peptide TFA salt. The molecular weight is determined byLC/MS (Found: [M+3H]³⁺=1638.4; Calculated [M+3H]³⁺=1638.53; Found MW(avg)=4912.2; Calc. MW (avg): 4912.58).

Example 2Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)AQ-Aib-DFVNWLLAQGPSSGAPPPS-NH₂(SEQID NO:6)

The structure of SEQ ID NO:6 is depicted below using the standard singleletter amino acid codes with the exceptions of residues D-Ala2, K17,Aib20, and Ser39, where the structures of these amino acid residues havebeen expanded:

The compound according to SEQ ID NO:6 is prepared substantially asdescribed by the procedures of Example 1, where instead the protecteddiacid is mono-OtBu-octadecanedioic acid (WuXi AppTec, Shanghai, China).The molecular weight is determined by LC/MS (Found: [M+3H]³⁺=1629.15;Calc. [M+3H]³⁺=1629.18; Found MW (avg)=4884.45; Calc. MW (avg)=4884.53).

Example 3Y-Aib-EGTFISDYSI-αMeL-LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-Aib-DFVE-4-Pal-LLEAGPSSGAPPPS-NH₂(SEQ ID NO:7)

The structure of SEQ ID NO:7 is depicted below using the standard singleletter amino acid codes with the exceptions of residues Aib2, αMeL13,K17, Aib20, 4-Pal25, and Ser39, where the structures of these amino acidresidues have been expanded:

The compound according to SEQ ID NO:7 is prepared substantially asdescribed by the procedures of Example 1. The molecular weight isdetermined by LC/MS (Found: [M+3H]³⁺=1662.52; Calc. [M 3H]³⁺=1662.55;Found MW (avg)=4984.55; Calc. MW (avg)=4984.64).

Example 4Y-Aib-EGTFISDYSI-αMeL-LD-Orn-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-Aib-DFVE-4-Pal-LLEAGPSSGAPPPS-NH₂(SEQ ID NO:8)

The structure of SEQ ID NO:8 is depicted below using the standard singleletter amino acid codes with the exceptions of residues Aib2, αMeL13,Orn16, K17, Aib20, 4-Pal25, and Ser39, where the structures of theseamino acid residues have been expanded:

The compound according to SEQ ID NO:8 is prepared substantially asdescribed by the procedures of Example 1. The molecular weight isdetermined by LC/MS (Found: [M+3H]³⁺=1657.82; Calc. [M+3H]³⁺=1657.87;Found MW (avg)=4970.46; Calc. MW (avg)=4970.62).

Example 5Y-Aib-EGTFISDYSI-αMeL-LDKIHQ-Aib-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂(SEQ ID NO:9)

The structure of SEQ ID NO:9 is depicted below using the standard singleletter amino acid codes with the exceptions of residues Aib2, αMeL13,Aib20, K26, and Ser39, where the structures of these amino acid residueshave been expanded:

The compound according to SEQ ID NO:9 is prepared substantially asdescribed by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is usedfor the lysine at position 26 rather than at position 17. The molecularweight is determined by LC/MS (Found: [M+3H]³⁺=1662.8; Calc.[M+3H]³⁺=1662.88; Found MW (avg)=4985.4; Calc. MW (avg)=4985.63).

Example 6Y-Aib-EGTFISDYSI-αMeL-LD-Orn-IHQ-Aib-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂(SEQID NO:10)

The structure of SEQ ID NO:10 is depicted below using the standardsingle letter amino acid codes with the exceptions of residues Aib2,αMeL13, Orn16, Aib20, K26, and Ser39, where the structures of theseamino acid residues have been expanded:

The compound according to SEQ ID NO:10 is prepared substantially asdescribed by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is usedfor the lysine at position 26 rather than at position 17. The molecularweight is determined by LC/MS (Found: [M+3H]³⁺=1658.1; Calc.[M+3H]³⁺=1658.20; Found: MW (avg)=4971.3; Calc. MW (avg)=4971.6).

Example 7Y-Aib-EGTFISDYSI-αMeL-LD-Orn-IHQ-Aib-EFV-(D-Glu)-YK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)LEGGPSSGAPPPS-NH₂(SEQID NO:11)

The structure of SEQ ID NO:11 is depicted below using the standardsingle letter amino acid codes with the exceptions of residues Aib2,αMeL13, Orn16, Aib20, D-Glu24, K26, and Ser39, where the structures ofthese amino acid residues have been expanded:

The compound according to SEQ ID NO:11 is prepared substantially asdescribed by the procedures of Example 1, where Fmoc-Lys(Mtt)-OH is usedfor the lysine at position 26 rather than at position 17 and theprotected diacid is mono-OtBu octadecanedioic acid (WuXi AppTec,Shanghai, China). The molecular weight is determined by LC/MS (Found:[M+3H]³⁺=1653.4; Calc. [M+3H]³⁺=1653.52; Found MW (avg)=4957.2; Calc. MW(avg)=4957.57).

The compounds according to Example 8 (SEQ ID NO:12) through Example 122(SEQ ID NO:126) are prepared substantially as described by theprocedures of Example 1.

Found SEQ Calculated (avg Example ID NO: Compound Name (avg) MW mw 8 12Y-(Aib)-EGTFISDYSILLDKK((2-[2-(2- 4926.61 4926.6Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)AQ-(Aib)-DFVNWLLAQGPSSGAPPPS-NH₂ 9 13 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4978.644978.4 Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVNWLLAQGPSSGAPPPS-NH₂ 10 14 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-4950.59 4950.4 Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)HQQDFVNWLLAGGPSSGAPPPS- NH₂ 11 15Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4955.61 4955.2Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)AQQDFVNWLLAQGPSSGAPPPS- NH₂ 12 16Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4884.53 4884.4Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)AQQDFVNWLLAGGPSSGAPPPS- NH₂ 13 17Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 5035.7 5035.2Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)HQQEFVNWLLAQGPSSGAPPPS- NH₂ 14 18Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 5036.68 5036.8Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)HQQDFVEWLLAQGPSSGAPPPS- NH₂ 15 19Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4998.63 4998.4Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)HQQDFVNYLLAQGPSSGAPPPS- NH₂ 16 20Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4899.54 4899.3Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₆—CO₂H)AQ-(Aib)-DFVEWLLAQGPSSGAPPPS-NH₂ 17 21 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-4942.56 4941.9 Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)AQ-(Aib)- DFVNWLLEQGPSSGAPPPS-NH₂ 18 22Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4846.48 4846.5Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)AQ-(Aib)-DFVN-(4- Pal)-LLAQGPSSGAPPPS-NH₂ 19 23Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4813.45 4813.2Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₆—CO₂H)AQ-(Aib)-DFVNWLLAGGPSSGAPPPS-NH₂ 20 24 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-4898.55 4898.4 Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)AQ-(Aib)- EFVNWLLAQGPSSGAPPPS-NH₂ 21 25Y-(Aib)-EGTFISDYSIALDKK((2-[2-(2- 4828.42 4827.9Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)HQ-(Aib)-DFVE-(4- Pal)-LLAGGPSSGAPPPS-NH₂ 22 26Y-(Aib)-EGTFISDYSILLDKK((2-[2-(2- 4870.5 4870.5Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)HQ-(Aib)-DFVE-(4- Pal)-LLAGGPSSGAPPPS-NH₂ 23 27Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4842.45 4842.3[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAGGPSSGAPPPS-NH₂ 24 28Y-(Aib)-EGTFISDYSI-(Aib)-LDKK((2-[2- 4842.45 4842.3(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAGGPSSGAPPPS-NH₂ 25 29Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4907.56 4907.1[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVNWLLAGGPSSGAPPPS-NH₂ 26 30 Y-(Aib)-EGT-αMeF(2F)- 4925.56 4925.4ISDYSIALDKK((2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- EFVNWLLAGGPSSGAPPPS-NH₂ 27 31Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4841.5 4841.4Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₆—CO₂H)AQ-(Aib)-EFVQWLLAGGPSSGAPPPS-NH₂ 28 32 Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2-4885.51 4885.2 Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)AQ-(Aib)- EFVNWLLEGGPSSGAPPPS-NH₂ 29 33Y-(D-Ala)-EGTFISDYSILLDKK((2-[2-(2- 4842.49 4842.45Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₆—CO₂H)AQ-(Aib)-EFVEWLLAGGPSSGAPPPS-NH₂ 30 34 Acetyl-(D-Tyr)-AEGT-αMeF(2F)- 4902.474902.0 ISDYSIALDKK ((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆— CO₂H)HQ-(Aib)-EFVNYLLAGGPSSGAPPPS-NH₂ 31 35 Y-(Aib)-EGTFISDYSILLDKK((2-[2-(2- 4884.534884.6 Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₆—CO₂H)HQ-(Aib)-DFVNYLLAAGPSSGAPPPS-NH₂ 32 36 Y-(D-Ala)-EGTLISDYSILLDKK((2-[2-(2-4850.51 4850.4 Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)AQ-(Aib)- DFVNWLLAQGPSSGAPPPS-NH₂ 33 37Acetyl-(D-Tyr)-AEGT-αMeF(2F)- 4988.56 4988.1ISDYSIALDKK((2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- EFVNYLLEGGPSSGAPPPS-NH₂ 34 38 Y-(Aib)-EGT-αMeF(2F)-4946.57 4946.4 ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)HQ-(Aib)-EFVNYLLATGPSSGAPPPS-NH₂ 35 39 Y-(Aib)-EGT-(αMeL)-ISDYSIALDKK((2- 4894.564894.2 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- EFVNYLLATGPSSGAPPPS-NH₂ 36 40Y-(Aib)-EGT-αMeF(2F)- 5005.59 5005.8 ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)HQ-(Aib)-DFVEYLLETGPSSGAPPPS-NH₂ 37 41 Y-(Aib)-EGT-αMeF(2F)- 4961.54 4961.4ISDYSIALDKK((2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- DFVEYLLEGGPSSGAPPPS-NH₂ 38 42 Y-(Aib)-EGT-αMeF(2F)-4990.58 4990.2 ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)HQ-(Aib)-DFVE-(4-Pal)-LLETGPSSGAPPPS-NH₂ 39 43 Y-(Aib)-EGT-αMeF(2F)- 4946.53 4946.4ISDYSIALDKK((2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE-(4-Pal)- LLEGGPSSGAPPPS-NH₂ 40 44Y-(Aib)-EGT-αMeF(2F)- 5028.63 5028.6 ISDYSIALDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)HQ-(Aib)-DFVEWLLETGPSSGAPPPS-NH₂ 41 45 Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4972.594972.5 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLETGPSSGAPPPS-NH₂ 42 46Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4999.61 4999.8[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLEQGPSSGAPPPS-NH₂ 43 47Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4942.56 4942.2[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLEAGPSSGAPPPS-NH₂ 44 48Y-(Aib)-EGT-(αMeF)-ISDYSIALDKK((2- 4928.54 4928.1[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLEGGPSSGAPPPS-NH₂ 45 49Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5014.67 5014.65[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLETGPSSGAPPPS-NH₂ 46 50Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5041.69 5041.35[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLEQGPSSGAPPPS-NH₂ 47 51Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4970.62 4970.4[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLEGGPSSGAPPPS-NH₂ 48 52Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4912.58 4912.8[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAGGPSSGAPPPS-NH₂ 49 53Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4884.51 4884.6[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAGGPSSGAPPPS-NH₂ 50 54Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 4926.61 4926.9LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLAGGPSSGAPPPS-NH₂ 51 55 Y-(Aib)-EGT-αMeF(2F)-ISDYSI-(αMeL)- 4944.6 4944.6LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLAGGPSSGAPPPS-NH₂ 52 56 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4926.61 4926.3[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVE- (4-Pal)-LLAGGPSSGAPPPS-NH₂ 53 57Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4927.59 4927.5[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVEYLLAGGPSSGAPPPS-NH₂ 54 58 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4950.634950.6 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- DFVEWLLAGGPSSGAPPPS-NH₂ 55 59Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4864.54 4864.8LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLAGGPSSGAPPPS-NH₂ 56 60 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4926.61 4926.6[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-D- (αMeF)-VE-(4-Pal)-LLAGGPSSGAPPPS- NH₂57 61 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4997.69 4997.5[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVE- (4-Pal)-LLAQGPSSGAPPPS-NH₂ 58 62Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4970.66 4970.4[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVE- (4-Pal)-LLATGPSSGAPPPS-NH₂ 59 63Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4940.63 4940.4[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVE- (4-Pal)-LLAAGPSSGAPPPS-NH₂ 60 64Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 5011.71 5011.5LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-EFVE-(4-Pal)-LLAQGPSSGAPPPS-NH₂ 61 65 Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 4984.69 4984.8LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-EFVE-(4-Pal)-LLATGPSSGAPPPS-NH₂ 62 66 Y-(Aib)-EGT-(αMeF)-ISDYSI-(αMeL)- 4954.66 4954.2LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-EFVE-(4-Pal)-LLAAGPSSGAPPPS-NH₂ 63 67 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5070.73 5070.9[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVEYLLEQGPSSGAPPPS-NH₂ 64 68 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5043.715043.9 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- EFVEYLLETGPSSGAPPPS-NH₂ 65 69Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5013.68 5013.9[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVEYLLEAGPSSGAPPPS-NH₂ 66 70 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5029.685029.2 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- DFVEYLLETGPSSGAPPPS-NH₂ 67 71Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5056.71 5056.8[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVEYLLEQGPSSGAPPPS-NH₂ 68 72 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4999.654999.2 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- DFVEYLLEAGPSSGAPPPS-NH₂ 69 73Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4985.63 4985.7[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVEYLLEGGPSSGAPPPS-NH₂ 70 74 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4956.634956.3 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLATGPSSGAPPPS-NH₂ 71 75Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5052.72 5052.6[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVEWLLETGPSSGAPPPS-NH₂ 72 76 Y-(Aib)-EGTLISDYSI-(αMeL)-LDKK((2- 5009.695009.7 [2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)- EFVEYLLETGPSSGAPPPS-NH₂ 73 77Y-(Aib)-EGTLISDYSI-(αMeL)-LDKK((2- 4965.64 4965.3[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ- Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-EFVEYLLEGGPSSGAPPPS-NH₂ 74 78 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4922.574922.4 LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLEGGPSSGAPPPS-NH₂ 75 79 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4966.63 4966.8LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLETGPSSGAPPP5-NH₂ 76 80 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)-LD- 4908.55 4908.6(Orn)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLEGGPSSGAPPPS-NH₂ 77 81 Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)-LD- 4952.6 4952.4(Orn)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLETGPSSGAPPP5-NH₂ 78 82 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4926.61 4926.3[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAAGPSSGAPPPS-NH₂ 79 83Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4983.66 4983.3[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAQGPSSGAPPPS-NH₂ 80 84Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5000.64 5000.4K((2-[2-(2-Amino-ethoxy)-ethoxy]- acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE-(4-Pal)-LLETGPSSGAPPP5- NH₂ 81 85Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5027.67 5027.7K((2-[2-(2-Amino-ethoxy)-ethoxy]- acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE-(4-Pal)-LLEQGPSSGAPPPS- NH₂ 82 86Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4956.59 4956.45K((2-[2-(2-Amino-ethoxy)-ethoxy]- acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE-(4-Pal)-LLEGGPSSGAPPPS- NH₂ 83 87Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4957.57 4957.8[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLATGPSSGAPPPS-NH₂ 84 88Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4984.6 4984.2[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAQGPSSGAPPPS-NH₂ 85 89Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4927.55 4927.2[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAAGPSSGAPPPS-NH₂ 86 90Y-(Aib)-EGTFISDYSI-(αMeL)-LDEK((2- 4913.52 4913.1[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLAGGPSSGAPPPS-NH₂ 87 91Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4942.56 4942.8K((2-[2-(2-Amino-ethoxy)-ethoxy]- acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)HQ-(Aib)-DFVE-(4-Pal)-LLEAGPSSGAPPPS- NH₂ 88 92Y-(Aib)-EGT-(Iva)-ISDYSI-(αMeL)- 4936.6 4936.2LDKK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ- (Aib)-DFVE-(4-Pal)-LLEAGPSSGAPPPS-NH₂ 89 93 Y-(Aib)-EGTLISDYSI-(αMeL)-LDKK((2- 4950.63 4950.6[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (4-Pal)-LLEAGPSSGAPPPS-NH₂ 90 94Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 5013.64 5013.6[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (αMeY)-LLEAGPSSGAPPPS-NH₂ 91 95Y-(Aib)-EGTFISDYSI-(αMeL)-LDKK((2- 4999.61 4999.5[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)HQ-(Aib)-DFVE- (αMeY)-LLEGGPSSGAPPPS-NH₂ 92 96Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 5056.71 5057.1(Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LEQGPSSGAPPPS-NH₂ 93 97Y-(D-Ala)-EGTFISDYSILLDKIAQ-(Aib)- 4884.53 4884.3DFVNWK((2-[2-(2-Amino-ethoxy)- ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)LAQGPSSGAPPPS-NH₂ 94 98 Y-(Aib)-EGT-αMeF(2F)- 4874.46 4874.4ISDYSIALDKIHQ-(Aib)-EFVNYK((2-[2- (2-Amino-ethoxy)-ethoxy]-acetyl)₂(γ-Glu)-CO—(CH₂)₁₆— CO₂H)LAGGPSSGAPPPS-NH₂ 95 99 Y-(Aib)-EGT-αMeF(2F)-4960.55 4960.8 ISDYSIALDKIHQ-(Aib)-EFVNYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂(γ- Glu)-CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 96 100 Y-(Aib)-EGTFISDYSIALDKIHQ-(Aib)- 4870.54870.2 EFVNYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LAGGPSSGAPPPS-NH₂ 97 101Y-(Aib)-EGT-(αMeF)-ISDYSIALDKIHQ- 4884.53 4884.6(Aib)-EFVNYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LAGGPSSGAPPPS-NH₂ 98 102Y-(Aib)-EGT-αMeF(2F)- 4902.52 4902.15 ISDYSIALDKIHQ-(Aib)-EFVNYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂(γ- Glu)-CO—(CH₂)₁₈—CO₂H)LAGGPSSGAPPPS-NH₂ 99 103 Y-(Aib)-EGT-αMeF(2F)- 4961.54 4961.4ISDYSIALDKIHQ-(Aib)-DFVEYK((2-[2- (2-Amino-ethoxy)-ethoxy]-acetyl)₂(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LEGGPSSGAPPPS-NH₂ 100 104Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 4984.64 4984.2(Aib)-EFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO— (CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 101105 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4971.6 4971.75IHQ-(Aib)-DFV-(D-Glu)-YK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 102 106 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4985.63 4985.1IHQ-(Aib)-EFV-(D-Glu)-YK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 103 107 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4943.55 4943.4IHQ-(Aib)-DFVEYK((2-[2-(2-Amino- ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₆—CO₂H)LEGGPSSGAPPPS-NH₂ 104 108Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4999.65 4999.2IHQ-(Aib)-EFVEYK((2-[2-(2-Amino- ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEAGPSSGAPPPS-NH₂ 105 109Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5029.68 5029.2IHQ-(Aib)-EFVEYK((2-[2-(2-Amino- ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LETGPSSGAPPPS-NH₂ 106 110Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 5056.71 5056.8IHQ-(Aib)-EFVEYK((2-[2-(2-Amino- ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEQGPSSGAPPPS-NH₂ 107 111Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4985.63 4985.4IHQ-(Aib)-EFVEYK((2-[2-(2-Amino- ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 108 112Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4956.59 4956.3IHQ-(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 109 113 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 4970.62 4970.4(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO— (CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 110114 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ- 4942.56 4942.2(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO— (CH₂)₁₆—CO₂H)LEGGPSSGAPPPS-NH₂ 111115 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4928.54 4928.4IHQ-(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₆—CO₂H)LEGGPSSGAPPPS-NH₂ 112 116 Y-(Aib)-EGT-αMeF(2F)- 4946.53 4946.4ISDYSIALDKIHQ-(Aib)-DFVE-(4-Pal)- K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LEGGPSSGAPPPS-NH₂ 113 117Y-(Aib)-EGT-αMeF(2F)- 4960.55 4960.8 ISDYSIALDKIHQ-(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]- acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEAGPSSGAPPPS-NH₂ 114 118 Y-(Aib)-EGT-αMeF(2F)- 5017.6 5017.2ISDYSIALDKIHQ-(Aib)-DFVE-(4-Pal)- K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H) LEQGPSSGAPPPS-NH₂ 115 119Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIRQ- 5004.63 5004.6(Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LEGGPSSGAPPPS-NH₂ 116 120Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)- 4990.61 4990.2IRQ-(Aib)-DFVEYK((2-[2-(2-Amino- ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 117 121 Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIHQ-4999.65 4999.8 (Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LEAGPSSGAPPPS-NH₂ 118 122Y-(Aib)-EGTFISDYSI-(αMeL)-LDKIRQ- 5018.66 5018.4(Aib)-DFVEYK((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LEAGPSSGAPPPS-NH₂ 119 123Y-(Aib)-EGT-αMeF(2F)- 4988.57 4988.4 ISDYSILLDKIHQ-(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]- acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEGGPSSGAPPPS-NH₂ 120 124 Y-(Aib)-EGT-αMeF(2F)- 5002.59 5002.5ISDYSILLDKIHQ-(Aib)-DFVE-(4-Pal)- K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈— CO₂H)LEAGPSSGAPPPS-NH₂ 121 125Y-(Aib)-EGT-αMeF(2F)- 5059.64 5059.8 ISDYSILLDKIHQ-(Aib)-DFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]- acetyl)₂-(γ-Glu)-CO—(CH₂)₁₈—CO₂H)LEQGPSSGAPPPS-NH₂ 122 126 Y-(Aib)-EGTFISDYSI-(αMeL)-LD-(Orn)-4984.64 4984.80 IHQ-(Aib)-EFVE-(4-Pal)-K((2-[2-(2-Amino-ethoxy)-ethoxy]-acetyl)₂-(γ-Glu)- CO—(CH₂)₁₈—CO₂H)LEAGPSSGAPPPS-NH₂

Binding Assays

Glucagon (referred to as Gcg or hGcg) is a Reference Standard preparedat Eli Lilly and Company. GLP-1(7-36)-NH₂ (referred to as GLP-1 orhGLP-1) is obtained from CPC Scientific (Sunnyvale, Calif., 97.2%purity, 100 μM aliquots in 100% DMSO). GIP(1-42)-NH₂ (referred to asGIP) is prepared at Lilly Research Laboratories using peptide synthesisand HPLC chromatography as described above (>80% purity, 100 μM aliquotsin 100% DMSO). [¹²1]-radiolabeled Gcg, GLP-1, or GIP is prepared using[¹²⁵I]-lactoperoxidase and obtained from Perkin Elmer (Boston, Mass.).

Stably transfected cell lines are prepared by subcloning receptor cDNAinto a pcDNA3 expression plasmid and transfected into human embryonickidney (HEK) 293 (hGcgR and hGLP-1R) or Chinese Hamster Ovary (CHO)(hGIPR) cells followed by selection with Geneticin (hGLP-1R and hGIPR)or hygromycin B (hGcgR).

Two methods are used for the preparation of crude cell membranes.

Method 1:

Frozen cell pellets are lysed on ice in hypotonic buffer containing 50mM Tris HCl, pH 7.5, and Roche Complete™ Protease Inhibitors with EDTA.The cell suspension is disrupted using a glass Potter-Elvehjemhomogenizer fitted with a Teflon® pestle for 25 strokes. The homogenateis centrifuged at 4° C. at 1100×g for 10 minutes. The supernatant iscollected and stored on ice while the pellets are resuspended inhomogenization buffer and rehomogenized as described above. Thehomogenate is centrifuged at 1100×g for 10 minutes. The secondsupernatant is combined with the first supernatant and centrifuged at35000×g for 1 hour at 4° C. The resulting membrane pellet is resuspendedin homogenization buffer containing protease inhibitors at approximately1 to 3 mg/mL, quick frozen in liquid nitrogen and stored as aliquots ina −80° C. freezer until use.

Method 2:

Frozen cell pellets are lysed on ice in hypotonic buffer containing 50mM Tris HCl, pH 7.5, 1 mM MgCl₂, Roche Complete™ EDTA-free ProteaseInhibitors and 25 units/mL DNAse I (Invitrogen). The cell suspension isdisrupted using a glass Potter-Elvehjem homogenizer fitted with aTeflon® pestle for 20 to 25 strokes. The homogenate is centrifuged at 4°C. at 1800×g for 15 minutes. The supernatant is collected and stored onice while the pellets are resuspended in homogenization buffer (withoutDNAse I) and rehomogenized as described above. The homogenate iscentrifuged at 1800×g for 15 minutes. The second supernatant is combinedwith the first supernatant and centrifuged an additional time at 1800×gfor 15 minutes. The overall supernatant is then centrifuged at 25000×gfor 30 minutes at 4° C. The resulting membrane pellet is resuspended inhomogenization buffer (without DNAse I) containing protease inhibitorsat approximately 1 to 3 mg/mL and stored as aliquots in a −80° C.freezer until use.

Binding Determination Methods

The equilibrium binding dissociation constants (K_(d)) for the variousreceptor/radioligand interactions are determined from homologouscompetition binding analysis instead of saturation binding due to highpropanol content in the [¹²⁵I] stock material. The K_(d) valuesdetermined for the receptor preparations were as follows: hGcgR (3.9nM), hGLP-1R (1.2 nM) and hGIPR (0.14 nM).

[¹²⁵I]-Glucagon Binding

The human Gcg receptor binding assays are performed using aScintillation Proximity Assay (SPA) format with wheat germ agglutinin(WGA) beads (Perkin Elmer). The binding buffer contains 25 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, 2.5mM CaCl₂), 1 mM MgCl₂, 0.1% (w/v) bacitracin (Research Products), 0.003%(w/v) Polyoxyethylenesorbitan monolaurate (TWEEN®-20), and RocheComplete™ Protease Inhibitors without EDTA. Peptides and Gcg are thawedand 3-fold serially diluted in 100% DMSO (10 point concentrationresponse curves). Next, 5 μL serially diluted compound or DMSO istransferred into Corning® 3632 clear bottom assay plates containing 45μL assay binding buffer or unlabeled Gcg control (non-specific bindingor NSB, at 1 μM final). Then, 50 μL [¹²⁵I]-Gcg (0.15 nM final), 50 μLhuman GcgR membranes (1.5 μg/well) and 50 μL of WGA SPA beads (80 to 150μg/well) are added with a Biotek Multiflo dispenser. Plates are sealedand mixed on a plate shaker (setting 6) for 1 minute and read with aPerkinElmer Trilux MicroBeta® scintillation counter after 12 hours ofincubation/settling time at room temperature. Final assay concentrationranges for peptides tested in response curves is typically 1150 nM to0.058 nM and for the control Gcg from 1000 nM to 0.05 nM.

[¹²⁵I]-GLP-1 Binding

The human GLP-1 receptor binding assay is performed using an SPA formatwith WGA beads. The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mMCaCl₂, 1 mM MgCl₂, 0.1% (w/v) bacitracin, 0.003% (w/v) TWEEN®-20, andRoche Complete™ Protease Inhibitors without EDTA. Peptides and GLP-1 arethawed and 3-fold serially diluted in 100% DMSO (10 point concentrationresponse curves). Next, 5 serially diluted compound or DMSO istransferred into Corning® 3632 clear bottom assay plates containing 45μL assay binding buffer or unlabeled GLP-1 control (non-specific bindingor NSB, at 0.25 μM final). Then, 50 μL [¹²⁵I]-GLP-1 (0.15 nM final), 50μL human GLP-1R membranes (0.5 μg/well and 50 μL of WGA SPA beads (100to 150 μg/well) are added with a Biotek Multiflo dispenser. Plates aresealed and mixed on a plate shaker (setting 6) for 1 minute and readwith a PerkinElmer Trilux MicroBeta® scintillation counter after 5 to 12hours of incubation/settling time at room temperature. Final assayconcentration ranges for peptides tested in response curves aretypically 1150 nM to 0.058 nM and for the control GLP-1, 250 nM to 0.013nM.

[¹²⁵I]-GIP Binding

The human GIP receptor binding assay is performed using an SPA formatwith WGA beads. The binding buffer contains 25 mM HEPES, pH 7.4, 2.5 mMCaCl₂, 1 mM MgCl₂, 0.1% (w/v) bacitracin, 0.003% (w/v) TWEEN®-20, andRoche Complete™ Protease Inhibitors without EDTA. Peptides and GIP arethawed and 3-fold serially diluted in 100% DMSO (10 point concentrationresponse curves). Next, 5 μL serially diluted compound or DMSO istransferred into Corning® 3632 clear bottom assay plates containing 45μL assay binding buffer or unlabeled GIP control (non-specific bindingor NSB, at 0.25 μM final). Then, 50 μL [¹²⁵I]-GIP (0.075-0.15 nM final),50 μL human GIPR membranes (3 μg/well) and 50 μL of WGA SPA beads (100to 150 μg/well) are added with a Biotek Multiflo dispenser. Plates aresealed and mixed on a plate shaker (setting 6) for 1 minute and readwith a PerkinElmer Trilux MicroBeta® scintillation counter after 2.5 to12 hours of incubation/settling time at room temperature. Final assayconcentration ranges for peptides tested in response curves is typically1150 to 0.058 nM or 115 nM to 0.0058 nM and for the control GIP, 250 nMto 0.013 nM.

Binding Assay Data Analysis

Raw CPM data for concentration curves of peptides, Gcg, GLP-1, or GIPare converted to percent inhibition by subtracting nonspecific binding(binding in the presence of excess unlabeled Gcg, GLP-1, or GIP,respectively) from the individual CPM values and dividing by the totalbinding signal, also corrected by subtracting nonspecific binding. Dataare analyzed using four-parameter (curve maximum, curve minimum, 1050,Hill slope) nonlinear regression routines (Genedata Screener, version12.0.4, Genedata AG, Basal, Switzerland). The affinity constant (K_(i))is calculated from the absolute 1050 value based upon the equationK_(i)=IC₅₀/(1+D/K_(d)) where D is the concentration of radioligand usedin the experiment, 1050 is the concentration causing 50% inhibition ofbinding and K_(d) is the equilibrium binding dissociation constant ofthe radioligand (described above). Values for K_(i) are reported as thegeometric mean, with error expressed as the standard error of the mean(SEM) and n is equal to the number of independent replicates (determinedin assays performed on different days). Geometric Means are calculatedas follows:Geometric Mean=10(Arithmetic Mean of Log Ki Values))

-   -   n=y/x means that only a subset of replicates (y) out of the        total number of replicates (x) is used to express the mean. SEM        is only calculated when y=2 or greater. Means are expressed as        geometric means with the standard error of the mean (SEM) and        the number of replicates (n) indicated in parentheses.

TABLE 1 In vitro Binding Affinity (K_(i)) of indicated Examples andcomparator molecules for human GcgR, GIPR, and GLP-1R in the presence of0.1% bacitracin. hGcgR hGIPR hGLP1R Example or Ki (nM) Ki (nM) Ki (nM)Comparator (SEM, n) (SEM, n) (SEM, n) hGIP-NH₂ 1150 0.125 1100 (18.3, n= 4) (0.00511,  (143, n = 4)  n = 319) hGlucagon 3.05 >2420 >4940(0.120, n = 457) (n = 3) (n = 5) hGLP-1 (7- >4590 >2300 0.785 36) NH₂ (n= 5) (n = 3) (0.0252, n = 489) 1 228 0.0667 57.8 (46.6, n = 5) (0.0566,n = 5) (16.7, n = 5) 2 379 0.0373 77.9  (218, n = 3) (0.000430, (20.2, n= 3) n = 3)  3 338 0.0525 381  (220, n = 8) (0.0215, n = 8)  (226, n =8) 4 431 0.0620 423  (253, n = 6) (0.0259, n = 6)  (235, n = 6) 5 6190.0771 >913  (140, n = 9) (0.0342, n = 9) (n = 9) 6 453 0.104 571  (125,n = 7) (0.0866, n = 7)  (368, n = 7) 7 >961 0.193 >913 (n = 5)  (0.157,n = 5) (n = 5) 8 101 0.027 38.1 9 >230 0.110 168 10 >230 0.159 60.7 11163 0.0584 85.9 12 134 0.0876 46.8 13 >230 0.237 >213 14 >230 0.460 >21315 >254 0.471 >236 16 549 0.0438 99.2 17 274 0.0571 106 18 463 0.0331158 19 335 0.0469 15.2 20 216 0.0477 79.8 21 >1530 1.46 801 22 >13800.219 388 23 323 0.0341 251 24 >960 0.254 729 25 101 0.0417 15.8 26 74.10.045 16.5 27 401 0.0354 23.2 28 86.8 0.0732 6.46 29 133 0.0569 22.7 30407 0.037 26.5 31 >959 0.0473 266 32 >959 0.0343 85.2 33 63.5 0.036931.2 34 143 0.034 201 35 >960 0.0351 177 36 201 0.0683 393 37 51.4 0.02683.9 38 182 0.0588 776 (59.8, n = 2) (0.0112, n = 2)  (134, n = 2) 3976.0 0.0444 235 (19.3, n = 2) (0.00439, n = 2)  (8.34, n = 2) 40 50.90.0594 302 41 758 0.143 >906 42 99.1 0.0392 563 43 285 0.0366 605  (121,n = 2) 44 50 0.059 209 45 660 0.0663 >909  (257, n = 3) (0.0241, n = 3)(n = 3) 46 435 0.0319 744 (89.6, n = 3) (0.00724, n = 3)  (58.2, n = 3)47 175 0.0452 320 (69.7, n = 3) (0.0127, n = 3) (77.8, n = 3) 48 2670.0498 211 49 >960 0.0512 596 50 23.4 0.0501 34.9 51 26.7 0.0386 54.1 52114 0.0372 128 53 152 0.0184 62.9 54 386 0.0326 49.9 55 >960 0.0331 26256 193 0.0487 530 57 422 0.0154 280 58 418 0.0324 459 (1.17, n = 2)(0.00694, n = 2)  (68.3, n = 2) 59 230 0.0148 109 60 26.2 0.0390 51.4 6180.4 0.0665 135 62 31.3 0.0414 47.6 63 185 0.0248 327 64 196 0.022 47765 279 0.0316 305 66 279 0.0866 326 68 290 0.0948 421 69 114 0.0406 14470 857 0.0421 570 71 102 0.0422 347 72 837 0.0594 437 73 292 0.036 11774 511 0.0287 395 75 >958 0.0626 >902 76 358 0.0293 263 77 >9580.0841 >905 (n = 2) (0.0207, n = 2) (n = 2) 78 505 0.0236 394 79 8880.0229 486 80 >958 0.0828 >906 (n = 2) (0.0284, n = 2) (n = 2) 81 6990.0374 714 (35.4, n = 2) (0.0106, n = 2) (39.8, n = 2) 82 305 0.0617 388 (112, n = 2) (0.00946, n = 2)   (140, n = 2) 83 >958 0.116 452 84 >9580.0584 325 85 800 0.0529 220 86 462 0.0533 108 87 576 0.0538 849 88 7880.0830 261 89 611 0.0720 294 (n = 3) (0.0169, n = 4) (72.9, n = 4) 90112 0.129 102 91 54.4 0.122 37.4 92 >958 0.0331 >902 93 >959 0.0430 >90994 114 0.0310 >909 95 85.0 0.0255 832 96 >960 0.232 >908 97 77.5 0.0249570 98 38.4 0.0195 311 99 174 0.0341 >901 100 572 0.0674 >913  (182, n =4) (0.0265, n = 3) (n = 3) 101 >961 0.139 >913 (n = 2) (0.0585, n = 2)(n = 2) 102 >961 0.164 >913 (n = 3)  (0.113, n = 3) (n = 3) 103 >9600.0405 >913 104 >960 0.134 >910 105 >960 0.174 >910 106 304 0.0918 >910107 347 0.0722 >911 108 >960 0.134 >910 109 >960 0.117 >908 110 >9600.339 >908 111 >960 0.0871 >908 112 257 0.0742 >908 113 821 0.108 >921(3.12, n = 4) (0.0287, n = 5) (n = 5) 114 >971 0.121 >921 (n = 3)(0.0496, n = 4) (n = 4) 115 373 0.0997 >912 (n = 2) (0.0472, n = 2) (n =2) 116 178 0.166 >912 (n = 2) (0.0753, n = 2) (n = 2) 117 685 0.0722 591118 NA 0.0696 >920 119 68.8 0.129 723 (n = 2) (0.0552, n = 2)  (119, n =2) 120 575 0.171 >912 (n = 2)  (0.231, n = 2) (n = 2) 121 94.90.103 >912 (n = 2) (0.0473, n = 2) (n = 2) 122 957 0.0311 >913

As demonstrated in Table 1, examples of the present invention are verypotent binders of the human GIPR, and have lower affinity for the GLP-1Rand GcgR.

cAMP Pharmacological Functional Assay in Presence of 0.1% Casein

A set of cAMP assays are conducted in HEK293 cells expressing the humanGLP-1 receptor (GLP-1R), glucose-dependent insulinotropic peptidereceptor (GIPR), or glucagon receptor (GcgR). Each receptorover-expressing cell line (20 μl) is treated with the test peptide inDMEM (Gibco Cat #31053) supplemented with 0.1% Casein (Sigma Cat#C4765), 250 μM IBMX, 1× GlutaMAX™ (Gibco Cat #35050), and 20 mM HEPES(HyClone Cat #SH30237.01) in a 20 μl assay volume. After incubating for60 minutes at room temperature, the resulting increase in intracellularcAMP is quantitatively determined using the CisBio cAMP Dynamic 2 HTRFAssay Kit (62AM4PEJ). The Lysis buffer containing cAMP-d2 conjugate (20μl) and the antibody anti-cAMP-Eu3+-Cryptate (20 μl) are then added todetermine the cAMP level. After incubating 60 minutes at roomtemperature, HTRF signal is detected with an Envision 2104 plate reader(PerkinElmer). Fluorescent emission at 620 nm and at 665 nm is measuredand the ratio between 620 nm and at 665 nm is calculated and then areconverted to nM cAMP per well using a cAMP standard curve. Dose responsecurves of compounds are plotted as the percentage of stimulationnormalized to minimum (buffer only) and maximum (maximum concentrationof each control ligand) values and analyzed using a four parameternon-linear regression fit with a variable slope (Genedata Screener 13).EC50 is the concentration of compound causing half-maximal simulation ina dose response curve. A relative EC₅₀ value is derived by non-linearregression analysis using the percent maximal response vs. theconcentration of peptide added, fitted to a four-parameter logisticequation.

Using Homogeneous Time Resolved Fluorescence methods, assays areconducted to determine the intrinsic potency of Example and comparatormolecules performed in the presence of casein (instead of serum albumin)as a nonspecific blocker, which does not interact with the fatty acidmoieties of the analyzed molecules.

Intracellular cAMP levels are determined by extrapolation using astandard curve. Dose response curves of compounds are plotted as thepercentage of stimulation normalized to minimum (buffer only) andmaximum (maximum concentration of each control ligand) values andanalyzed using a four-parameter non-linear regression fit with avariable slope (Genedata Screener 13). EC₅₀ is the concentration ofcompound causing half-maximal simulation in a dose response curve. Eachrelative EC50 value for the geometric mean calculation is determinedfrom a curve fitting.

Concentration response curves of compounds are plotted as the percentageof stimulation normalized to minimum (buffer only) and maximum (maximumconcentration of each control ligand) values and analyzed using afour-parameter non-linear regression fit with a variable slope (GenedataScreener 13). EC50 is the concentration of compound causing half-maximalsimulation in a dose response curve. The EC₅₀ summary statistics arecomputed as follows: Geometric mean:GM=10^((arithmetic mean of log) ¹⁰ ^(transformed EC) ⁵⁰ ^(values)). Thestandard error of the mean is reported:SEM=geometric mean×(standard deviation of log₁₀ transformed EC₅₀values/square root of the # of runs)×log_(e) of 10.

-   -   The log transform accounts for the EC₅₀ values falling on a        multiplicative, rather than an arithmetic scale.

Each time the assay is performed, the test peptides are run plus thenative ligands GIP, GLP-1, and glucagon, buffer only as baseline(minimum) and the highest concentration of the respective GIP, GLP-1,and glucagon standard is used as maximum for calculations. Forillustration, as shown by Example 1, the peptide is tested in 8 runs ofthe hGIPR cAMP assay. For avoidance of doubt, hGIP amide, hGLP-1 amide,and glucagon EC50 in Table 2 are illustrative of geometric mean valuesfrom a series of 18 assay values, and values will vary each day comparedto the zero buffer. Accordingly, each Example will use the geometricmean of those values to normalize the Example assay runs.

TABLE 2 Functional activation of hGcgR, hGIPR, and hGLP-1R in thepresence of 0.1% Casein. hGcgR cAMP hGIPR cAMP hGLP1R cAMP Example orRel EC50 nM Rel EC₅₀ nM Rel EC₅₀ nM Comparator (SEM, n) (SEM, n) (SEM,n) hGIP-NH₂ >5000 0.122 >500 (n = 5) (0.00449, (n = 3) n = 494)hGlucagon 0.0116 9.79 (0.000315, (1.83, n = 3) n = 306)hGLP-1(7-36) >500 0.0549 NH₂ (n = 4) (0.00149, n = 490) 1 5750.0476 >5000  (369, n = 4) (0.0253, n = 8) (n = 5) 2 608 0.0123 >5000 (n = 11) (0.00751, n = 15)  (n = 11) 3 1250 0.0178 >5000  (398, n = 6)(0.00680, n = 9)  (n = 6) 4 1690 0.0182 >5000 (n = 5) (0.00783, n = 5) (n = 5) 5 >5000 0.0148 >5000 (n = 5) (0.00434, n = 5)  (n = 5) 6 >50000.0176 >5000 (n = 4) (0.00714, n = 14) (n = 4) 7 >5000 0.0219 >5000 (n =2) (0.00294, n = 2)  (n = 2) 8 295 0.0268 >5000 (62.9, n = 2) (0.000153,(n = 2) n = 3) 9 >5000 0.165 >100 10 >5000 0.611 >100 11 445 0.0738 >10012 >5000 0.0911 >100 13 >5000 0.129 >100 14 >5000 0.340 >100 15 >50000.282 >100 16 >5000 0.00937 >5000 (n = 2) (0.00210, n = 2)  (n = 2)17 >5000 0.0135 >5000 (n = 2) (0.00227, n = 2)  (n = 2) 18 >50000.00656 >5000 (n = 2) (0.00228, n = 2)  (n = 2) 19 >5000 0.00992 >5000(n = 2) (0.00148, n = 3)  (n = 2) 20 >5000 0.00933 >5000 (n = 2)(0.00112, n = 3)  (n = 2) 21 >5000 0.291 >5000 (n = 3) (0.0766, n = 4)(n = 3) 22 1250 0.0345 >5000  (487, n = 3) (0.00839, n = 4)  (n = 3)23 >5000 0.0192 206 (n = 5) (0.00529, n = 4)  (13.8, n = 4) 24 >50000.0699 >5000 (n = 2) (0.0155, n = 3) (n = 2) 25 >5000 0.0155 8.39 (n =2) (0.00237, n = 3)  (2.46, n = 4) 26 >5000 0.00991 5.69 (n = 2)(0.00277, n = 2)  (2.48, n = 4) 27 >5000 0.0132 >5000 (n = 2) (0.000266,(n = 2) n = 2) 28 >5000 0.0118 >5000 (n = 2) (0.00124, n = 2)  (n = 2)29 >5000 0.00679 >5000 (n = 4) (0.00193, n = 4)  (n = 4) 30 >50000.00572 >5000 (n = 4) (0.00160, n = 4)  (n = 4) 31 1750 0.0148 >5000 (886, n = 3) (0.00426, n = 3)  (n = 3) 32 >5000 0.00469 >5000 (n = 2)(0.00199, n = 3)  (n = 2) 33 >5000 0.0551 >5000 34 913 0.0177 462  (586,n = 2) (0.00478, n = 2)   (321, n = 2) 35 >5000 0.0125 >5000 36 >50000.0543 1080 37 >5000 0.0360 61.1 38 >5000 0.0368 1390 39 >5000 0.01123330 40 >5000 0.0330 2330 41 >5000 0.0281 1800 42 >5000 0.0091 81543 >5000 0.0154 1020 44 >5000 0.0084 1180 45 1690 0.0490 >5000 (n = 2)(0.000420, (n = 2) n = 2) 46 863 0.0294 >5000 (n = 2)  (0.0166, n = 2)(n = 2) 47 757 0.0234 >5000  (492, n = 2) (0.00230, n = 2)  (n = 2) 481360 0.0166 >5000 49 2860 0.0156 >5000  (620, n = 6) (0.00569, n = 7) (n = 6) 50 371.0 0.0212 41.0 51 308.0 0.0166 24.7 52 337.0 0.0194 >500053 344 0.0194 >5000  (136, n = 2) (0.00603, n = 3)  (n = 2) 54 >50000.0540 >5000 55 >5000 0.0170 >5000 (n = 2) 56 >5000 0.0169 >5000 (n = 2)57 886 0.0177 >5000  (430, n = 2) (0.00819, n = 3)  (n = 2) 58 >50000.0183 >5000 (0.00544, n = 2)  59 >5000 0.0202 >5000 60 >5000 0.0369 7161 >5000 0.0167 192 62 >5000 0.0116 58.2 63 1170 0.0398 >5000 64 30700.0448 >5000 65 850 0.0346 >5000 66 >5000 0.0786 >5000 67 >50000.0627 >5000 68 3030 0.0768 >5000 (n = 2) (n = 2) 69 803 0.0302 >5000 (237, n = 2) (0.00976, n = 2)  70 3560 0.0254 >5000 71 581 0.0721 >500072 >5000 0.0182 >5000 73 >5000 0.0151 >5000 74 627 0.0167 >5000 75 21700.0182 >5000 76 1200 0.0154 >5000 77 2660 0.0265 >5000 78 30000.0125 >5000 (n = 2) (0.00185, n = 2)  (n = 2) 79 >5000 0.0316 >500080 >5000 0.0777 >5000 (n = 2) (0.0223, n = 2) (n = 2) 81 >50000.0282 >5000 (n = 2) (0.00791, n = 2)  (n = 2) 82 3790 0.0391 >5000 (n =2) (0.00658, n = 2)  (n = 2) 83 >5000 0.0432 >5000 84 >5000 0.0340 >500085 >5000 0.0359 >5000 86 >5000 0.0300 >5000 87 >5000 0.0107 >5000 881670 0.0031 >5000 89 >5000 0.00687 >5000 (n = 2) (0.00245, n = 2)  (n =2) 90 >5000 0.0272 >5000 91 289 0.0321 530 92 >5000 0.0191 >5000 (n = 2)(0.00110, n = 2)  (n = 2) 93 >5000 0.00482 884 (n = 2) (0.000315, (n =2) n = 2) 94 >5000 0.00436 1090 (n = 4) (0.00186, n = 4)   (442, n = 4)95 >5000 0.0272 >5000 (0.0110, n = 2) 96 >5000 0.0251 >5000 97 >50000.0090 2510 98 >5000 0.00718 649 (n = 2) (0.00331, n = 3)   (369, n = 2)99 >5000 0.00454 >5000 (0.000120, n = 2) 100 >5000 0.0224 >5000101 >5000 0.0396 >5000 (n = 2) (0.00639, n = 2)  (n = 2) 102 >50000.0166 >5000 (n = 2) (0.00337, n = 2)  (n = 2) 103 >5000 0.0129 >5000 (n= 2) (0.00901, n = 2)  (n = 2) 104 >5000 0.0165 >5000 105 >50000.0179 >5000 106 >5000 0.0140 >5000 107 >5000 0.0199 >5000 108 >50000.0088 >5000 109 >5000 0.0113 >5000 110 >5000 0.0071 >5000 111 >50000.0065 >5000 112 >5000 0.0041 >5000 113 >5000 00.0142 >5000 (n = 3)(0.0108, n = 3) (n = 3) 114 >5000 0.0075 >5000 115 >5000 0.0372 >5000116 >5000 0.0280 >5000 117 >5000 0.0617 >5000 118 >5000 0.0611 >5000119 >5000 0.0220 >5000 120 >5000 0.0228 >5000 121 >5000 0.0196 >5000122 >5000 0.0100 >5000

As demonstrated by data in Table 2, Example compounds of the presentinvention are very potent stimulating cAMP from human GIPR in thepresence of 0.1% casein.

In Vivo Studies

Pharmacokinetics in Male Cynomolgus Monkeys

The pharmacokinetics of select Examples are evaluated following a singlesubcutaneous administration of 50 nmol/kg to male cynomolgus monkeys.Blood samples are collected over 504 hours and resulting individualplasma concentrations are used to calculate pharmacokinetic parameters.Peptide plasma (K₃ EDTA) concentrations are determined using a qualifiedLC/MS method that measured the intact mass of the compound. Each peptideand an analog as an internal standard are extracted from 100% cynomolgusmonkey plasma using a protein precipitation method. Instruments arecombined for LC/MS detection. Mean pharmacokinetic parameters are shownin Table 3.

TABLE 3 Mean Pharmacokinetic Parameters of peptides Following a SingleSubcutaneous Administration of 50 nmol/kg to Male Cynomolgus Monkeys.C_(max)/Dose AUC_(Inf)/Dose T_(1/2) T_(max) (kg*nmol/ (hr*kg*nmol/L/CL/F Example (hr) (hr) L/nmol) nmol) (mL/hr/kg) 1 71.1 24 7.20 786 1.272 51.8 6 6.92 624 1.61 3 88.8 60 12.1 1764 0.57 4 124 6 8.71 1387 0.72 5128 120 12.0 2262 0.44 6 129 6 7.83 1382 0.77 7 109 9 10.1 1603 0.63Abbreviations: T_(1/2) = half-life, T_(max) = time to maximalconcentration, C_(max)/dose = maximal plasma concentration divided bydose, AUC_(Inf)/Dose = AUC_(Inf) divided by dose, CL/F =clearance/bioavailability. Notes: Data are the mean, where n = 2/group.

As seen in Table 3, results from this study for Example peptides testedare consistent with an extended pharmacokinetic profile.

Pharmacokinetics in Male Sprague Dawley Rats Following SubcutaneousAdministration

The pharmacokinetics of select Examples are evaluated following a singlesubcutaneous (SC) administration of 100 nmol/kg to male Sprague Dawleyrats. Blood samples are collected over 168 hours following SCadministration. Pharmacokinetic parameters are calculated usingindividual plasma concentrations. A qualified LC/MS method that measuresthe intact mass of the Example is used to determine plasma (1(3 EDTA)concentrations. Each peptide and an analog as an internal standard areextracted from 100% rat plasma using a protein precipitation method.Instruments are combined for LC/MS detection. Mean pharmacokineticparameters for the Examples are shown in Table 4.

TABLE 4 Mean (+/−SD) Pharmacokinetic Parameters of peptides Following aSingle Subcutaneous Administration of 100 nmol/kg to Male Sprague Dawleyrats. AUC_(Inf)/Dose C_(max)/Dose (hr*kg*nmol/ CL/F Example T_(1/2) (hr)T_(max) (hr) (kg*nmol/L/nmol) L/nmol) (mL/hr/kg) 1 37.4 24 3.62 280 3.572 26.9 12 5.47 304 3.30 3 24.9 12 3.03 130 7.67 5 27.1 24 4.61 239 4.206 34.8 24 4.07 271 3.69 Abbreviations: T_(1/2) = half-life, T_(max) =time to maximal concentration, C_(max)/Dose = maximal plasmaconcentration divided by dose, AUC_(Inf)/Dose = AUC_(Inf) divided bydose, CL/F = clearance/bioavailability. Notes: Data are the mean, wheren = 3/group, except for example 3, where the data is from n = 1 animal.

As seen in table 4, results from this study using these Example peptidesare consistent with an extended pharmacokinetic profile.

In Vivo Effect on Insulin Secretion in Male Wistar Rats

Male Wistar rats with femoral artery and femoral vein canulas (Envigo,Indianapolis, Ind.) (280-320 grams) are single-housed in polycarbonatecages with filter tops. Rats maintained on a 12:12 h light-dark cycle(lights on at 6:00 A.M.) at 21° C. and receive food and deionized waterad libitum. Rats are randomized by body weight and dosed 1.5 mL/kgsubcutaneously (s.c.) at doses of 0.3, 1.0, 3, 10, 30, and 100 nmol/kg16 hours prior to glucose administration then fasted. Animals areweighed and anesthetized with sodium pentobarbital dosedintraperitoneally (i.p.) (65 mg/kg, 30 mg/mL). At time 0, a blood sampleis collected into EDTA tubes after which glucose is administeredintravenously (i.v.) (0.5 mg/kg, 5 mL/kg). Blood samples are collectedfor glucose and insulin levels at 2, 4, 6, 10, 20 and 30 minpost-intravenous administration of glucose. Plasma glucose levels aredetermined using a clinical chemistry analyzer. Plasma insulin isdetermined using an electrochemiluminescence assay (Meso Scale,Gaithersburg, Md.). Glucose and insulin AUC are examined compared to thevehicle control with n=5 animals per group. Results are presented(SEM)(N).

TABLE 5 The effect of Example compounds on insulin secretion duringintravenous glucose tolerance test. Dose (nmol/kg, s.c.) Example 0.0 0.31.0 3.0 10 30 100 3 38.3 43.3 45.1 53.3 64.9 116.3 133.6 (8.8)(5)(0.8)(5) (8.8)(5) (6.5)(5) (9.3)(5) (28.6)(4) (16.1)(5) 5 35.9 36.2 45.380.6 122.0 144 212.7 (7.5)(5) (4.4)(5) (6.1)(5) (6.4)(5) (5.9)(5)(16.4)(5) (19.4)(5) 6 39.5 34.7 54.2 75.8 100.2 135.4 166.5 (2.2)(5)(3.6)(5) (6.8)(5) (10.3)(5) (15.6)(5) (22.9)(5) (21.6)(5)

The data provided by Table 5 demonstrate a dose dependent increase ininsulin secretion.

TABLE 6 ivGTT Insulin Secretion shown by the following data: Insulinsecretion (ivGTT) Example (ED₅₀, nmol/kg) (SEM, n) 3 17.1 (n = 1) 5 18.4(n = 1) 6 12.9 (n = 1)

-   -   The data provided by Table 6 demonstrate dose dependent increase        in insulin secretion.

Immunogenicity Assessment of the Compounds of Examples 1, 2, 3, 5, and 6

The purpose of this study is to determine the relative potential forclinical immunogenicity of Example compounds 1, 2, 3, 5, and 6.

Methods:

CD4+ T Cell Assay: The CD4+ T cell assay is used to compare thecompounds of Examples 1, 2, 3, 5, and 6 for a potential to induce animmune response in vivo according to methods known in the art (see,e.g., Jones et al. (2004) J. Interferon Cytokine Res. 24:560-572; andJones et al. (2005) J. Thromb. Haemost. 3:991-1000), where an assessmentof clinically tested monoclonal antibodies and peptides shows somedegree of correlation between T cell proliferation observed in vitro andimmunogenicity in the clinic. Protein therapeutics that induce less than30% positive response in the CD4+ T cell proliferation assay areassociated with a low risk of immunogenicity. Briefly, to assess thepropensity fora clinical immunogenic response to the compounds ofExamples 1, 2, 3, 5, and 6, CD8+ T cell-depleted peripheral bloodmononuclear cells (PBMCs) are prepared and labeled withcarboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen) froma cohort of 10 healthy donors with diverse human leukocyte antigen (HLA)class II haplotypes. Each donor is tested in triplicate with 2.0 mLmedia control, keyhole limpet hemocyanin (KLH; 0.33 μM), and thecompounds of Examples 1, 2, 3, 5, and 6 (0.33 μM). Cultures areincubated for 7 days at 37° C. with 5% CO₂. On day 7, samples areanalyzed by flow cytometry using a BD LSR II Fortessa (Becton Dickinson;Franklin Lakes, N.J.), equipped with a high throughput sampler (HTS).Data is analyzed using FlowJo® Software (FlowJo, LLC/TreeStar; Ashland,Oreg.).

Results and Discussion

All donors produce a positive T cell response against KLH (100%).Analysis of the frequency and magnitude of the CD4+ T cell response forExample compounds is shown in Table 7.

TABLE 7 CD4+ T Cell Responses for Example compounds and Positive Control(KLH). Median Response Example or % Donor Response Strength in positiveComparator (n = 10) donors (CDI) KLH 100%  164.285 (n = 10) Example 1 0%NA (n = 0) Example 2 10%  2.69 (n = 1) Example 3 0% NA (n = 0) Example 50% NA (n = 0) Example 6 0% NA (n = 0)

-   -   Cell Division Index (“CDI”): proportion of divided CD4+ T cells        to the total number of CD4+ T cells in stimulated versus        unstimulated samples.

These data show that the frequency of positive CD4+ T cell response(CDI>2.5) was low for the tested Example compounds, and the magnitude ofthe response in the one positive donor from the Example 2 group was low(CDI<3). Thus, based on this assay, these compounds have a low risk ofimmunogenicity.

Amino Acid Sequences SEQ ID NO: 1 GIP 1-42 (Human)YAEGTFISDYSIAMDKIHQQDFVNWLLAQKGKKNDWKHNITQ SEQ ID NO: 2GLP-1 (7-36) amide (Human) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH₂SEQ ID NO: 3 Glucagon (Human) HSQGTFTSDYSKYLDSRRAQDFVQWLMNT SEQ ID NO: 4Z₁X₁X₂EGTX₆ISDYSIX₁₃LDX₁₆X₁₇X₁₈QX₂₀X₂₁X₂₂VX₂₄X₂₅ X₂₆LX₂₈X₂₉GPSSGAPPPSZ₂

We claim:
 1. A compound comprising SEQ ID NO:7, or a pharmaceuticallyacceptable salt thereof.
 2. A method for treating a condition selectedfrom the group consisting of diabetes mellitus, obesity, and metabolicsyndrome, comprising administering to a patient in need thereof, aneffective amount of a compound, or a pharmaceutically acceptable saltthereof, as claimed by claim
 1. 3. A pharmaceutical compositioncomprising the compound, or a pharmaceutically acceptable salt thereof,as claimed by claim 1, and at least one pharmaceutically acceptablecarrier, diluent, or excipient.